The overall goal of this project is to identify changes in cellular gene expression which cooperate with HPV in the progression of HPV immortalized cells to malignancy. The steps in the conversion of human epithelial cells to tumorigenicity will be studied. Cofactors identified as important in epidemiology projects of this program will be examined in transformation and tumor induction assays for effects on cells expressing HPV genes and for direct effects on viral gene expression. cDNA libraries will be constructed from immortalized, transformed and tumor cells and differentially expressed genes identified by subtractive hybridization. Human tumor tissue will be screened using probes derived from these experiments. The development and significance of chromosomal changes will be examined by cytogenetic analysis to develop an understanding of the genetic instability found in HPV-immortalized cells. One aim of this analysis will be to identify loci of tumor suppressor genes involved in HPV-associated tumor cells. The factors contributing to the blocks in terminal differentiation in HPV immortalized cells will be examined in the organotypic cell culture system.